PROJECT SUMMARY/ABSTRACT The brain's control of our thoughts, feelings, and behaviors stems from neural circuits, which perform logical operations based on the temporal patterns of neural activity and the connectivity of the neurons as the circuit traverses the brain. Recent studies have produced many strategies for visualizing and controlling a circuit's neural activity. In some cases, specialized microscopy systems have enabled imaging of entire brain volumes on the timescale of neural activity and during behavior, enabling the reconstruction of neural circuitry at cellular resolution. However, recording neural activity from whole brain volumes to reconstruct neural circuitry is a significant challenge when working with larger brains, less specialized microscopes, or stimuli and behaviors that are not conducive to concurrent imaging. In these situations, strategies that enable permanent recording of neural activity during defined time windows for later readout would be highly beneficial. In this proposal, we describe an approach for permanent recording of neural activity using Ca2+-dependent enzymatic activation of genetically encoded substrates. We focus our efforts on the Tobacco Etch Virus (TEV) protease because it does not have endogenous substrates in vertebrate systems, it has been employed in living neurons, it has been extensively re-engineered to record interactions between diverse binding partners, and its split fragments dissociate in the absence of reconstituting binding partners. We propose a strategy for engineering a Ca2+-dependent TEV protease that involves a split version of the enzyme with each half attached to the Ca2+-binding partners calmodulin and M13. These TEV fragments are designed so that in the absence of neural activity they will remain separate and inactive, but in the presence of neural activity and high Ca2+, the association of calmodulin and M13 will permit TEV reconstitution and enzyme activity. Once active, the split TEV protease can cleave a variety of genetically encoded substrates that link TEV protease activity with transcription of a desired gene or direct creation of a fluorophore. Thus, using this strategy will enable users to control neurons activated by a given stimulus or behavior (e.g., by expressing optogenetic tools in response to TEV activity) or to visualize neurons active during distinct epochs (e.g., by expressing a photoactivatable mOrange in response to one stimulus or behavior, photoconverting the mOrange, and then recording the second stimulus). Finally, the split TEV protease and substrate interactions can be controlled with modular optogenetics tools to provide precise temporal control over the time period of recording.